Biocontrol of multidrug resistant pathogens isolated from fish farms using silver nanoparticles combined with hydrogen peroxide insight to its modulatory effect

This study was divided into two parts. The first part involved the isolation, and detection of the prevalence and antimicrobial resistance profile of Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio species from Nile tilapia fish and marine aquatic water. One hundred freshly dead Nile tilapia fish were collected from freshwater aquaculture fish farms located in Al-Abbassah district, Sharkia Governorate, and 100 samples of marine aquatic water were collected from fish farms in Port Said. The second part of the study focused on determining the in vitro inhibitory effect of dual-combination of AgNPs-H2O2 on bacterial growth and its down regulatory effect on crucial virulence factors using RT-PCR. The highest levels of A. hydrophila and P. aeruginosa were detected in 43%, and 34% of Nile tilapia fish samples, respectively. Meanwhile, the highest level of Vibrio species was found in 37% of marine water samples. Additionally, most of the isolated A. hydrophila, P. aeruginosa and Vibrio species exhibited a multi-drug resistance profile. The MIC and MBC results indicated a bactericidal effect of AgNPs-H2O2. Furthermore, a transcriptional modulation effect of AgNPs-H2O2 on the virulence-associated genes resulted in a significant down-regulation of aerA, exoU, and trh genes in A. hydrophila, P. aeruginosa, and Vibrio spp., respectively. The findings of this study suggest the effectiveness of AgNPs-H2O2 against drug resistant pathogens related to aquaculture.


expression, Silver nanoparticles
The aquaculture industry has been growing rapidly in recent years to meet the global demand for seafood.Unfortunately, it is also prone to fish and shell fish related diseases caused by pathogenic microorganisms resulting in significant economic losses 1 .Water is a precious resource, and its usage in aquaculture places a heavy burden on it.Unconsumed food excretory byproducts, chemicals, and antibiotics can all lead to bacterial, fungal, and viral illnesses in the ponds.The typical remedy for this is constant water changes, which not only consumes hundreds of cubic meters of water per day (depending on the size of the pond) but also pollutes the surrounding environment with dirty effluent 2,3 .Of particular importance in aquaculture are the bacteria Pseudomonas species; Aeromonas species and Vibrio species, which can cause disease in fish 4 .One of the most virulent Aeromonas species; A. hydrophila, is capable of causing significant issues for Egypt's freshwater fish farming 5 .Pseudomonas aeruginosa, a gram-negative, rod-shaped, obligate aerobic bacterium, can infect fish, particularly in harsh environmental conditions, leading to ulcer-type disorders.Furthermore, Pseudomonas aeruginosa, the primary cause of nosocomial infections, can be fatal in immunocompromised individuals 6 .Vibrio species are ubiquitous foodborne pathogens found in surface waters and are associated with food poisoning 7 .They are abundant in estuarine-marine and

Bacteriological analysis
The water samples were concentrated on nitrocellulose membrane filters (0.45-μm Isolation of A. hydrophila P. aeruginosa and Vibrio spp. was conducted following a protocol previously established 5,29 .In summary, a loopful from the spleen, kidney, and liver of each collected fish was enriched in tryptic soy broth (TSB, Oxoid®, USA) tubes and then incubated at 37 °C for 18-24 h.To isolate typical yellow A. hydrophila colonies, a loopful was sub-cultured by streaking on Rimler-Shott's medium (HiMedia, India) with novobiocin (Oxoid®, USA) supplement, and incubated for 24 h at 37 °C.For the isolation of typical fluorescent P. aeruginosa colonies, a loopful was sub-cultured by streaking on Pseudomonas F agar, and incubated for 24 h at 37 °C.The isolation of Vibrio spp. was carried out following a previously established protocol 30 .
The water samples were concentrated on nitrocellulose membrane filters (0.45-μm pore size; Millipore) by passing 100 mL through the filter using the membrane filtration technique.The filters were then placed onto alkaline peptone water (APW) and incubated for 24 h at 37 °C to enrich the growth of the Vibrio species.A loopful of culture from APW was streaked onto CHROMagar™ Vibrio (CHROMagar, Paris, France), and incubated at 37 °C for 24 h.Mauve, green-blue to turquoise blue, and creamy colonies representing V. parahaemolyticus, V. vulnificus, and V. alginolyticus respectively were picked up, and preserved.

Molecular detection and genotypic characterization of A. hydrophila P. aeruginosa and Vibrio spp.
Five isolates of each microbe (A.hydrophila, P. aeruginosa and Vibrio spp.) were selected for molecular detection in the Biotechnology Unit, Animal Health Research Institute, Zagazig Branch, Egypt.Presumptive colonies were grown overnight on Tryptone Soy Broth (Oxoid, USA).Bacterial DNA was extracted using a QIAamp DNA Mini kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's instructions.Polymerase chain reaction (PCR) amplifications were performed using oligonucleotide primers specified in Table S1.These included the aerA, act, and ast genes of A. hydrophila; toxA, exoS, and exoU genes of P. aeruginosa, and tdh, toxR, trh, genes of Vibrio species along with flaE, hsp, and collagenase genes of V. parahaemolyticus, V. vulnificus, and V. alginolyticus respectively.The reference strains (control positive) used in this study were Aeromonas hydrophila ATCC® 35654™*, Pseudomonas aeruginosa ATCC® 27853™*, Vibrio parahaemolyticus ATCC® 17802™*, Vibrio vulnificus ATCC® 27562™*, and Vibrio alginolyticus ATCC® 17749™*.

Antimicrobial activities of AgNPs-H2O2
Bacterial strains that exhibited the highest levels of antimicrobial resistance profiles were chosen to test the antimicrobial effectiveness of AgNPs-H2O2 against them.These strains included five multidrug-resistant A. hydrophila strains, four extensively drug-resistant P. aeruginosa strains, and one pan drug-resistant P. aeruginosa strain, as well as five extensively drug-resistant Vibrio species.The Agar Well Diffusion Method was utilized to assess the antibacterial properties of AgNPs-H2O2 as follows: Bacterial cultures were cultivated in sterile saline and adjusted to an optical density of 0.5 Macfarland (1.5 × 10 8 CFU/mL).The bacterial suspension was evenly spread on Mueller Hinton (MH) agar (Oxoid Ltd., England) using a sterile cotton swab.Wells (8 mm) were created in each inoculated agar plate and approximately 10 µL of AgNPs-H2O2 at a concentration of 10% (v/v) was added into each well.Sterile water was used as a negative control for bacterial growth instead of AgNPs-H2O2.The agar plates were then placed in the incubator at 37 °C for 24 h.The experiments were conducted in triplicate, and the antimicrobial activity was determined by measuring the diameter of the inhibition zone (mm).
The effectiveness of AgNPs-H2O2 against the selected bacterial strains was evaluated using Minimum Inhibitory concentration (MIC) following the procedure described previously described 37 .The MIC was determined according to CLSI guidelines 38 in a 96-well microtiter plate with 100 µL of a twofold serial dilution of commercial AgNPs-H2O2 in Muller Hinton broth (MHB) ranging from 32 to 0.0625 µg/mL, and bacterial suspension adjusted to 5 × 10 5 CFU/mL pipetted into the wells.Sterile Muller Hinton broth without NPs served as a negative control, and inoculated Muller Hinton broth without NPs served as positive control.The microtiter plate was then incubated for 24 h at 37 °C.The MIC value was considered the lowest concentration of a particular antibacterial agent that could inhibit bacterial growth.
To determine the Minimum Bactericidal Concentration (MBC), 10 μL of bacterial suspension starting from the MIC onwards (equal to or two concentrations higher than the MIC) were dropped on MH agar and incubated as described above.The lowest concentration of each antimicrobial that killed 99.9% of bacterial growth was considered the MBC 39 .Tolerance levels were determined using the formula: MBC/MIC 40 , and the agent is considered bactericidal when the MBC/MIC ratio is ≤ 4 41 .The sub-inhibitory concentration was determined as the highest concentration of antimicrobial that showed no effect on survivability and no growth inhibition.

Modulatory effect of AgNPs-H2O2
A reverse transcriptase quantitative PCR (RT-qPCR) analysis was conducted to investigate the modulatory effect of sub-inhibitory concentrations of AgNPs-H2O2, on the expression of aerA, act, and ast genes of A. hydrophila, toxA, exoS, and exoU genes of P. aeruginosa, and tdh, toxR, and trh genes for Vibrio species.Bacterial cultures were grown in the presence of AgNPs-H2O2 or without treatment as a reference control.Cells were harvested by centrifugation and cell pellet was mixed with 200 μL of RNA protect Bacteria Reagent (Qiagen, Hilden, Germany).Total RNA was extracted using the QIAamp RNeasy Mini kit (Qiagen, Germany, GmbH) following the manufacturer's protocol.Residual DNA was removed by DNase digestion column using the RNase-Free DNase Set (Qiagen) protocol.RNA purity and concentration were determined using Nanodrop measurement.Real-time PCR amplification reactions were prepared in a final volume of 20 µL containing 10 µL of the 2× HERA SYBR® Green RT-qPCR Master Mix (Willowfort, UK), 1 µL of RT Enzyme Mix (20×), 1 µL of each primer at a concentration of 20 pmol, 2 µL of RNase-and DNase-free water, and 5 µL of RNA template.Quantitative Real-Time PCR (qPCR) was carried out using primers for aerA, act, and ast genes of A. hydrophila, toxA, exoS, and exoU genes of P. aeruginosa, and tdh, toxR and trh, genes of Vibrio species, and amplification conditions were set using a Real time PCR machine (StepOne Plus, Thermo Fisher).Results were analyzed using the comparative cycle threshold method (ΔΔCT method), and normalized against 16S rRNA as the endogenous control as previously described 42 .

Statistical analysis
Each experiment was carried out at least in triplicate, and all data were presented as mean +/− standard deviation (SD).Analysis of statistical significance was performed by one-way analysis of variance (ANOVA) and the post hoc Tukey test (p < 0.05).All analysis was conducted in SAS 9.4 for Windows 64-bit from SAS Institute (Cary, NC), and graphical outputs were generated by GraphPad Prism software (version 8; GraphPad Software Inc.).

Antimicrobial resistance profiles
The in vitro antimicrobial susceptibilities of A. hydrophila, P. aeruginosa, and Vibrio species against antimicrobial agents are summarized in Tables S2, S3, and S4.The PDR, XDR, and MDR patterns were reported among the analyzed isolates (Fig. 6, Table 2).The MDR profiles were significantly increased among the A. hydrophila isolates with 90.69% (39 out of 43) exhibiting this resistant pattern.However, only 2% and 6%of the analyzed isolates showed SDR and DDR pattern respectively.
Considering Vibrio species, the multidrug resistant pattern was detected among most isolates (81%; 30 out of 37) with a higher prevalence of V. parahaemolyticus (50%;15 isolates) followed by V. vulnificus, and V. alginolyticus with 9 and 6 isolates (30%, and 20%) respectively.However, only 7 isolates showed an extensively drug resistant pattern (18.9%).(2 µg/mL) were detected in 40% of the tested bacterial species including 2 strains of A. hydrophila, 3 strains of Vibrio species and 1 strain of P. aeruginosa.The MBC was noted at 4 µg/mL with a tolerance level of 2 indicating the bactericidal effect of AgNPs-H 2 O 2 .The MIC results illustrated that all tested Vibrio species were more susceptible to AgNPs-H 2 O 2 than the other tested A. hydrophila, and P. aeruginosa, with a lower concentration

Transcriptional modulatory effect of AgNPs-H 2 O 2
To investigate the mechanism by which AgNPs-H 2 O 2 inhibit bacterial growth, real-time q RT-PCR was used to determine the expression levels of act, aerA, and ast genes in 5 multidrug resistant A. hydrophila isolates, toxA, exoS, and exoU genes in 5 P. aeruginosa isolates (4XDR, and 1PDR), and tdh, toxR, and trh in 5 extensively drug resistant Vibrio species reported here (Fig. 9).Data analysis indicated that all tested isolates showed downregulation of the tested genes upon treatment with sub-inhibitory concentration of AgNPs-H 2 O 2 compared to the untreated control isolates.The result indicated that A. hydrophila isolates treated with AgNPs-H 2 O 2 had the most significant down-regulation of the aerA gene by 0.17-0.29-foldchange.The suppression of exoU gene

Discussion
Aquaculture is essential for providing a reliable source of animal protein for human consumption in rapidly growing coastal nations like Egypt 43 .This supply is at risk from uncontrollable bacterial diseases, particularly from virulent and drug resistant pathogens such as A. hydrophila, P. aeruginosa and Vibrio spp.In this study, the prevalence of A. hydrophila in freshly deceased Nile tilapia was found to be 43%.Previous research has shown similar rates of A. hydrophila in Oreochromis niloticus [44][45][46][47] , while lower rates have also been reported [48][49][50] .The prevalence of P. aeruginosa in this study was consistent with previous findings with some studies reporting lower rates [51][52][53] .Previous studies have shown a higher prevalence of V. parahaemolyticus in farm water compared to this study [54][55][56][57] , while the isolation rate of V. vulnificus was similar to previous report 58 .The differences in prevalence  www.nature.com/scientificreports/rates of isolated bacteria could be due to factors such as the number of samples examined, variations in host susceptibility, geographical distribution, salt level, sampling period, and other environmental factors 59,60 .Increasing resistance to major antibiotics is an emerging problem in pathogenic bacteria found in the aquatic environment including fish and pond water.Therefore, investigating of the drug resistance profiles is essential to prevent the spread of antibiotic-resistant pathogens and search for better alternatives.Multidrug-resistant (MDR) bacteria have become a global concern in recent decades posing a serious threat to human, animal and fish health 61 .In this study A. hydrophila isolates were MDR in 90.69% of cases.This level of resistance was lower than previously reported 62 , while a lower prevalence was also detected 63 .Meanwhile, the resistance profile of Pseudomonas isolates in this study showed a 52.9% MDR rates with a mean MAR index between 0.125 and 1 and an average of 0.48 similar to previous findings 64 .Regarding Vibrio isolates, 81% were found to be MDR, with most isolates displaying multiple MAR indices ranging from 0.42 to 0.86 and an average of 0.57.This suggests contamination from hazardous sources and acquired genetic resistance leading to public health hazards 65 .Similarly, a high MAR index for V. parahaemolyticus isolates was reported previously 66 .The use of nanotechnology to modify their potential effects by regulating their size has been used as a biological alternative control strategy [67][68][69][70] .In this study, we evaluated the in vitro antimicrobial efficacy, and transcriptional modulatory effect of AgNPs-H2O2 against MDR microorganisms.The obtained findings showed a notable inhibition zone of AgNPs-H2O2 against A. hydrophila strains, P. aeruginosa and Vibrio species.This observation is supported by a previous study that described a pronounced antibacterial effect of AgNPs against isolated Vibrio species 71 .Previous studies also stated an effective MIC of AgNPs against P. aeruginosa 37 , A. hydrophila, and Vibrio species 72,73 .Corroborating these findings, we found that the MIC value of AgNPs-H2O2 against the examined isolates ranged from 0.5 to 8 µg/mL with tolerance level of 2 indicating the bactericidal effect of AgNPs-H2O2.Cytotonic heat-stable enterotoxins (ast), cytotoxic enterotoxin (act) and aerolysin (aer A) genes are the major virulence factor of A. hydrophila that exhibit hemolytic and cytolytic properties and causes cell death, depending on destroying the cell membrane permeability causing osmotic lysis 74,75 .Additionally, the toxA gene of P. aeruginosa inhibits the biosynthesis of host cell proteins 76 .The hemolytic exoenzyme encoded by the exoS gene causes tissue destruction and helps in bacterial dissemination 77 , and the exoenzyme-U encoded by the exoU gene is considered a marker of P. aeruginosa invasiveness as it is the most cytotoxic enzyme among type III secretion proteins 78 .Furthermore, many crucial proteins are commonly used to assess the pathogenicity of Vibrio isolates such as two hemolysins: thermostable direct hemolysin (tdh) and the tdh-related hemolysin (trh) 57,79 , as well as the toxR gene which regulates the expression of virulence factors and is used to categorize all isolates as pathogenic strains 80 .Thus, we investigated the transcriptional effect of AgNPs-H2O2 on crucial virulence factors of A. hydrophila, P. aeruginosa, and Vibrio species.This study revealed that A. hydrophila isolates treated with AgNPs-H2O2 showed the most significant downregulation of the aerA gene by 0.17-0.29-foldchange.Additionally, the suppression of exoU gene expression in P. aeruginosa isolates was more pounced with 0.09-0.21-foldchange.Meanwhile, the trh gene expression level was the most affected among the tested Vibrio species with 0.029-0.21-foldchange.This finding is constituent with previous studies that have reported the inhibitory effect of silver nanoparticles on the expression of virulence factors in multidrug-resistant Pseudomonas aeruginosa strains 81 .Furthermore, previous research has also documented the significant downregulation of virulence genes in VTEC O157:H7 in response to H2O2 82 .
The antimicrobial activity of the AgNPs-H2O2 (Silver Nanoparticles-Hydrogen Peroxide) combination arises from a synergistic effect between the two components and the breakdown of the mechanisms involved: 1. Silver Nanoparticles (AgNPs) cause direct contact and membrane disruption: AgNPs can physically interact with the bacterial cell wall, causing disruptions and increased permeability.This leakage of essential cellular components leads to cell death.Additionally, AgNPs can interact with oxygen and water molecules within the bacteria, promoting the formation of Reactive Oxygen Species (ROS) like superoxide radicals and hydrogen peroxide.These ROS damage vital cellular components such as proteins, DNA, and lipids 83 .2. Hydrogen Peroxide (H2O2) induces direct oxidative stress: H2O2 itself can act as a mild oxidizing agent, damaging cellular components to a certain extent 84 .
Thus, the combination of AgNPs and H2O2 can have a synergistic effect on ROS generation.AgNPs can catalyze the decomposition of H2O2 into hydroxyl radicals (OH•), which are even more potent ROS compared to hydrogen peroxide itself.These highly reactive hydroxyl radicals cause extensive damage to cellular components within the bacteria.In addition, H2O2 might also help improve the penetration of AgNPs into the bacterial cells, further enhancing their antimicrobial activity 85 .
Overall, the AgNPs-H2O2 combination disrupts bacterial cell membranes, generates high levels of ROS, leading to oxidative stress and damage to essential cellular components and can be more effective against a broader range of bacteria compared to using AgNPs or H2O2 alone.
This finding could be explained by a higher bactericidal action of more than 100 times due to the Fentonlike reaction between silver nanoparticles and hydrogen peroxide resulting in the formation of hydroxyl groups (-OH) the most potent biologically active ROS 86 .As an alternate antibacterial strategy, this combination is anticipated to provide a number of benefits: (a) the surface chemistry alteration of silver nanoparticles by conjugation of certain moieties, allowing for the selective targeting of bacterial cells and greatly reducing toxicity 87 .(b) Producing silver ions (Ag+), which have antibacterial activity of their own 88 .(c) Silver ions can block the catalase enzyme, which is generated by certain bacteria to neutralize hydrogen peroxide 89 .
In summary, the present study highlights the in vitro inhibitory effect of the dual combination of AgNPs-H2O2 on bacterial growth and the down regulatory effect on the crucial virulence factors of A. hydrophila, P. aeruginosa, and Vibrio species, making them excellent candidates for targeted drug delivery.

Figure 1 .
Figure 1.Prevalence of A. hydrophila, and P. aeruginosa species isolated from freshwater fishes, and Vibrio species from marine water including; V. parahaemolyticus, V. vulnificus, and V. alginolyticus.

2 Five
https://doi.org/10.1038/s41598-024-58349-4www.nature.com/scientificreports/Antibacterial effect of AgNPs-H 2 O A. hydrophila strains categorized as MDR (n = 5), five P. aeruginosa strains categorized as XDR (n = 4), and PDR (n = 1), and five XDR Vibrio species (n = 5) were selected and screened for the antibacterial properties of AgNPs-H 2 O 2 using the agar well diffusion method, Minimum Inhibitory Concentration (MIC) and minimum bactericidal concentration (MBC).The inhibitory zones were presented in Fig.7.A notable inhibition zone of 35-40 mm was observed with a 10% concentration of AgNPs-H 2 O 2 against A. hydrophila strains, while the inhibition zone observed among tested P. aeruginosa was 42-47 mm.The inhibitory activity value ranged from 35 to 45 mm for vibrio species.Notably, the effect of AgNPs-H 2 O 2 against Vibrio species showed variable susceptibility as V. parahaemolyticus was less susceptible to AgNPs-H 2 O 2 compared with V. vulnificus, and V. alginolyticus.The Minimum Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC) values were presented in Fig.8.The MIC of all tested A. hydrophila, P. aeruginosa, and Vibrio species showed that AgNPs-H 2 O 2 has an inhibitory effect on bacterial growth (0.5 to 8 µg/mL).The MIC values of AgNPs-H 2 O 2

Figure 7 .
Figure 7.The inhibitory effect of AgNPs-H 2 O 2 on A. hydrophila, P. aeruginosa, and Vibrio species growth.Antimicrobial susceptibility testing using the agar well diffusion method with representative images is shown in which AgNPs-H 2 O 2 was used at concentration of 10% (v/v).The antibacterial activity was measured by the inhibition zone in millimeters (mm).The experiments were done in triplicate and each column represented the mean ± SEM.

Figure 8 .
Figure 8. Minimum inhibitory concentration (MIC) of AgNPs-H 2 O 2 among A. hydrophila, P. aeruginosa, and Vibrio species growth in compared with untreated positive control, and un-inoculated negative control.Antimicrobial susceptibility testing using the broth microdilution method with representative images are shown in which MIC was measured by 32-0.0625μg/mL.SIC; sub-inhibitory concentration.MBC; minimum bactericidal concentration.

Figure 9 .
Figure 9. Relative expression of A. hydrophilaact, aerA, and ast genes (A), P. aeruginosa, toxA, exoS, and exoU genes (B), and Vibrio species tdh, toxR, and trh genes (C) upon treatment with AgNPs-H 2 O 2 .The fold change was determined by qRT-PCR, calculated using the ΔΔ CT method and normalized comparatively to 16S rRNA expression.The experiments were done in triplicate and each column represented the mean ± SEM.

Table 1 .
Prevalence of A. hydrophila, and P. aeruginosa species isolated from freshwater fishes, and Vibrio species from marine water including; V. parahaemolyticus, V. vulnificus, and V. alginolyticus..